PROJECT SUMMARY/ABSTRACT Cell-derived extracellular microvesicles (MVs) have recently emerged as a well-preserved evolutionary mechanism of hepatic cell-to-cell communication. The main functions of MVs are to signal target cells through specific interactions and to transfer gene products. Human liver stem cells (HLSCs) and mesenchymal stem cells (MSCs) were extensively investigated for their reparative, regenerative and immunomodulatory properties. We have made the following key observations regarding the cellular mechanisms of ncRNA gene expression in mesenchymal and hepatic stem cell derived microvesicles: (a) The biological action of microvesicles required their miR-181 and let-7 miRNA-dependent incorporation into hepatic cells. (b) In vivo, microvesicles accelerated the morphologic and functional recovery of CCl4- induced acute liver injury (ALI) and alcoholic liver injury (ALD) in SCID mice by inducing proliferation/anti-senescence effects of hepatic cells. (c) Stem cell derived MVs shuttle a specific subset of cellular ncRNA, such as miR-181, let-7, uc.189 and uc.338 that are associated with the stemness phenotypes and anti-apoptosis/anti-senescence potentials. (d) In human ALD liver tissues, miR-181b and let-7a are silenced whereas uc.189 and uc.338 are up-regulated. Based on the compelling data, we propose the central hypothesis that ncRNAs in stem cell derived MVs contribute to the recovery of alcoholic liver injury through anti-apoptosis and anti-senescence effects in hepatic cells. To test this hypothesis, we have established techniques for ncRNA and gene manipulation, functional investigation and interaction analysis, and generated animal models of acute and alcoholic liver injury (NIAAA model). Our long-term objective is to identify and isolate stem cell derived microvesicles and characterize their functional properties in tissue repair during alcoholic liver injury. In this application, we propose the systematic investigation of stemness dependent ncRNAs as markers in stem cell derived MVs, which possess therapeutic potential for alcoholic liver injury. We will address our central hypothesis by focusing on the following specific aims: 1) To identify functional, stemness regulated ncRNAs involved in tissue repair-related cellular functions in hepatic cells during alcoholic liver injury. In this study, we will characterize the interactions between miR-181/let-7 and uc.338/uc.189 family members and define the role of stemness dependent ncRNA signaling in ethanol treated hepatocytes with or without stem cell derived MVs and human ALD liver tissues through flow cytometry analysis, immunoblots and real-time PCR. 2) To determine the effects of stemness related ncRNA enriched microvesicles on accelerating the morphologic and functional recovery of acute and alcoholic liver injury in SCID/C57 mice with or without immunosuppression in vivo. Therapeutic effects of microvesicles derived from stem cells will be evaluated. New information on the mechanisms of ncRNA in stem cell derived microvesicles in ALD will be obtained. Meanwhile, the fundamental new knowledge acquired about regulation of tissue repair by stem cell derived microvesicles is expected to advance the general field of stem cell biology.